EP0345021B1

EP0345021B1 - Poultry mycoplasma antigens and recombinant vectors containing the gene as well as diagnostics and vaccines utilizing the same

Info

Publication number: EP0345021B1
Application number: EP89305441A
Authority: EP
Inventors: Kazumi Kodama; Shuji Saito; Noboru Yanagida; Kouichi Kamogawa; Yoshikazu Iritani; Shigemi Aoyama
Original assignee: Shionogi and Co Ltd; Nippon Zeon Co Ltd
Current assignee: Zeon Corp
Priority date: 1988-06-02
Filing date: 1989-05-31
Publication date: 1994-04-27
Anticipated expiration: 2009-05-31
Also published as: JP2670145B2; AU623657B2; CA1339260C; EP0345021A2; AU3594089A; US5621076A; EP0345021A3; DE68914883D1; US5766594A; KR910000177A; DE68914883T2; JPH02111795A

Description

The present invention relates to infection of poultry, and to methods and substances for diagnosing and vaccinating against said Mycoplasma gallisepticum infection.
Mycoplasma gallisepticum infectious disease that is one of the most serious infections on poultry such as chickens, is characterized by chronic respiratory disturbance accompanied by inflammation of the air sac in a chicken. A chicken infected with Mycoplasma gallisepticum as a pathogen develops very slight symptoms and rarely comes to death. However, when infected with Mycoplasma gallisepticum, an egg-laying rate and a hatching rate of eggs produced by infected chickens are markedly reduced. As the result, shipping of eggs and egg-laying chickens are decreased, resulting in the considerable economic loss. In addition, Mycoplasma gallisepticum infection induces the reduction in immunity so that chickens are liable to suffer from other infectious diseases to cause complication of severe infectious diseases. Furthermore, Mycoplasma gallisepticum is known to be a pathogen of infectious paranasal sinusitis in turkeys.
Efforts have been hitherto made to prevent poultry Mycoplasma gallisepticum infections using antibiotics or by vaccination and to establish pathogen-free poultry. However, administration of antibiotics involves defects that bacilli resistant to Mycoplasma would appear and Mycoplasma would readily proliferate after discontinuation of administration of antibiotics. On the other hand, prophylaxis by vaccination using attenuated Mycoplasma might rather cause opportunistic infections with other pathogens, or Mycoplasma gallisepticum infections. As stated above, it is highly difficult to establish and maintain a group of poultry free of infections with Mycoplasma.
Turning to determination whether or not poultry is infected with Mycoplasma gallisepticum, it has been judged by taking as a measure if serum collected from poultry could inhibit proliferation of Mycoplasma to form a characteristic inhibition zone on PPLO agar plate including glucose and donor horse serum. However, the method involves shortcomings that 3 to 7 days are required to judge whether or not a clear inhibition zone is formed; during the period, infection tends to be spread over the whole group of poultry.
As a result of extensive investigations to solve the drawbacks in the prior art, the present inventors have discovered polypeptides derived from Mycoplasma gallisepticum and having antigenicity of Mycoplasma gallisepticum and further found that antisera, derived from the polypeptides using said polypeptides as antigens, can prevent growth of Mycoplasma gallisepticum and, the polypeptides are expected to be useful as poultry vaccines capable of preventing Mycoplasma gallisepticum infections and are useful as poultry diagnostics for Mycoplasma gallisepticum infections. The present invention has thus come to be accomplished.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there are provided polypeptides which cause an antigen-antibody reaction with anti-Mycoplasma gallisepticum poultry sera.
According to a second aspect of the present invention, there is provided a gene encoding the polypeptides.
According to a third aspect of the present invention, there is provided a recombinant vector in which the gene has been integrated.
According to a forth aspect of the present invention, there is provided a host transformed or transfected by the recombinant vector.
According to a fifth aspect of the present invention, there is provided a poultry vaccine for Mycoplasma gallisepticum infections comprising the polypeptides as an effective ingredient.
Further according to a sixth aspect of the present invention, there is provided a poultry diagnostic for Mycoplasma gallisepticum infection comprising the polypeptides as an effective ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1(a)-1 to 1(f)-2 shows amino acid sequences of polypeptides MG-1, MG-2, MG-3, MG-7, MG-8 and MG-9 exhibiting antigenicity of Mycoplasma gallisepticum and base sequences of M-1, M-2, M-3, M-7, M-8 and M-9 encoding the polypeptides. Fig. 2(a) and 2(b) similarly shows an amino acid sequence of polypeptide TMG-1 and a base sequence of TM-1 encoding the polypeptide. Fig. 3-1 to 3-4 indicate restriction enzyme cleavage maps of DNA fragments encoding the polypeptides in accordance with the present invention. Figs. 4, 5 and 6 indicate procedures for producing antigen protein plasmids in accordance with the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the first aspect of the present invention, the polypeptides are the one that cause an antigen-antibody reaction with poultry sera infected with Mycoplasma gallisepticum.
Specific examples include DNA fragments derived from Mycoplasma gallisepticum and polypeptides that can be produced by fragments having restriction enzyme cleavage maps shown in Fig. 3, (a) through (v). More specifically, the polypeptides are as shown in Table 1 and exemplified by about 30 kilodaltons of polypeptide MG-1 derived from cloned antigen DNA M-1, about 1 kilodalton of polypeptide MG-2 derived from cloned antigen DNA M-2, about 30 kilodaltons of polypeptide MG-3 derived from cloned antigen DNA M-3, about 55 kilodaltons of polypeptide MG-4 derived from cloned antigen DNA M-4, about 1 kilodalton of polypeptide MG-5 derived from cloned antigen DNA M-5, about 32 kilodaltons of polypeptide MG-6 derived from cloned antigen DNA M-6, about 35 kilodaltons of polypeptide MG-7 derived from cloned antigen DNA M-7, about 35 kilodaltons of polypeptide MG-8 derived from cloned antigen DNA M-8, about 35 kilodaltons of polypeptide MG-9 derived from cloned antigen DNA M-9, about 55 kilodaltons of polypeptide MG-10 derived from cloned antigen DNA M-10, about 46 kilodaltons of polypeptide MG-11 derived from cloned antigen DNA M-11, about 15 kilodaltons of polypeptide MG-12 derived from cloned antigen DNA M-12, about 29 kilodaltons of polypeptide MG-13 derived from cloned antigen DNA M-13, about 15 kilodaltons of polypeptide MG-14 derived from cloned antigen DNA M-14, about 79 kilodaltons of polypeptide MG-15 derived from cloned antigen DNA M-15, about 15 kilodaltons of polypeptide MG-16 derived from cloned antigen DNA M-16, about 55 kilodaltons of polypeptide MG-17 derived from cloned antigen DNA M-17, about 49 kilodaltons of polypeptide MG-18 derived from cloned antigen DNA M-18, about 32 kilodaltons of polypeptide MG-19 derived from cloned antigen DNA M-19, about 35 kilodaltons of polypeptide MG-20 derived from cloned antigen DNA M-20, about 9 kilodaltons of polypeptide MG-21 derived from cloned antigen DNA M-21, about 38 kilodaltons of polypeptide MG-22 derived from cloned antigen DNA M-22, etc. The polypeptide is further exemplified by a polypeptide having amino acid sequence containing the amino acid sequence from one of these polypeptides and having the same amino acid sequence as the polypeptide expressed in Mycoplasma gallisepticum. The polypeptide is also exemplified by fused proteins having as C-terminus an amino acid sequence of MG-1, MG-2, MG-3, MG-7, MG-8, MG-9 shown in Fig. 1 (a)-1 through (f)-2 or TMG-1 shown in Fig. 2(a) and 2(b) and containing, as stabilizing protein, bacteria-derived enzyme proteins such as β-galactosidase, β-lactamase, etc. at the N-terminus thereof. Other polypeptides can also be converted into fused proteins with bacteria-derived enzyme proteins.
The polypeptides which are concerned with the first aspect of the present invention can be obtained by using the host (relating to the forth aspect of the invention) transformed or transfected by the recombinant vector that is concerned with the third aspect of the invention.
The recombinant vector described above can be obtained by integrating a Mycoplasma gallisepticum-derived DNA fragment into an expression vector in a conventional manner.
Sources for collecting the DNA fragment may be any one so long as they belong to Mycoplasma gallisepticum. Specific examples include S6 strain (ATCC 15302), PG31 (ATCC 19610) and the like.
Specific examples of the DNA fragment used for recombination are DNA fragment encoding the amino acid sequence shown in Fig. 1(a)-1 and Fig. 1(a)-2 (for example, 705 base pairs from 1 to 705 in Fig. 1(a)-1 and Fig. 1(a)-2), DNA fragment encoding the amino acid sequence shown in Fig. 1(b) (for example, 30 base pairs from 1 to 30 in Fig. 1(b)), DNA fragment encoding the amino acid sequence shown in Fig. 1(c) (for example, 657 base pairs from 1 to 657 in Fig. 1(c)), DNA fragment encoding the amino acid sequence shown in Fig. 1(d) (for example, 549 base pairs from 1 to 549 in Fig. 1(d)), DNA fragment encoding the amino acid sequence shown in Fig. 1(e) (for example, 531 base pairs from 1 to 531 in Fig. 1(e)), DNA fragment encoding the amino acid sequence shown in Fig. 1(f)-1 and Fig. 1(f)-2 (for example, 927 base pairs from 1 to 927 in Fig. 1(f)-1 and Fig. 1(f)-2); and DNA fragments added at the upstream of the 5' end thereof with a DNA fragment encoding enzyme protein, etc. of bacteria in combination with the reading frame. In addition thereto, the DNA fragment is also exemplified by DNA fragments derived from Mycoplasma gallisepticum such as M-4, M-5, M-6, M-10, M-11, M-12, M-13, M-14, M-15, M-16, M-17, M-18, M-19, M-20, M-21, M-22, etc. Restriction enzyme cleavage maps of these DNA fragments and their lengths are shown in Fig. 3. Furthermore, the DNA fragment is exemplified by the one, encoding the amino acid sequence of that having the same amino acid sequence as that of the polypeptide expressed in Mycoplasma gallisepticum and containing the amino acid sequence of these antigen polypeptide, etc. (for example, 783 base pairs from 40 to 822 in Fig. 2), given from genomic DNA of Mycoplasma gallisepticum in such a manner well known to one skilled in the art as the hybridization technique using these DNA fragments as probes.
The vector which is used to construct the recombinant vector is not particularly limited, however, specific examples include plasmids such as pUC8, pUC9, pUC10, pUC11, pUC18, pUC19, pBR322, pBR325, pBR327, pDR540, pDR720, and the like; phages such as λgt11, λgt10, λEMBL3, λEMBL4, Charon 4A and the like.
The method for inserting the DNA fragment described above into these vectors to produce recombinant vectors may be performed in a manner conventional to one skilled in the art. For example, the vector is cleaved with a restriction enzyme and ligated with the DNA fragments described above either directly or via synthetic linker, under control of a suitable expression regulatory sequence.
As the expression regulatory sequence used, those may be mentioned; ℓac promoter operator, trp promoter, tac promoter, ℓpp promoter, P_L promoter, amyE promoter, Gaℓ7 promoter, PGK promoter, ADH promoter, etc.
In producing the recombinant vector for the purpose of expressing these polypeptides derived from Mycoplasma, techniques for producing a recombinant vector by once integrating the aforesaid DNA fragment into a suitable vector and then carrying out subcloning, is well known to one skilled in the art. These subcloned DNA fragments are excised with an appropriate restriction enzyme and ligated under control of the expression regulatory sequence described above. Thus, the recombinant vector capable of producing the polypeptide can be produced.
The vector which is used for the subcloning is not critical but specific examples include plasmids such as pUC8, pUC9, pUC10, pUC11, pUC18, pUC19, pBR322, pBR325, pBR327, pDR540, pDR720, pUB110, pIJ702, YEp13, YEp24, YCp19, YCp50, and the like.
Using the obtained recombinant vector, a variety of appropriate hosts are transformed to micro-organisms that can produce the polypeptides capable of expressing antigenicity of Mycoplasma gallisepticum, a part of the polypeptides or a fused protein containing said polypeptides.
The appropriate host used herein can be chosen taking into account adaptability to expression vector, stability of the products, etc. Specific examples are genus Escherichia (for example, Escherichia coli), genus Bacillus (for example, Bacillus subtilis, Bacillus sphaericus, etc.), Actinomyces, Saccharomyces, etc. The host transformed with an appropriate expression vector can be cultured and proliferated under suitable culture conditions well known to one skilled in the art.
Upon production of the polypeptide, conditions for inducing the action of expression regulation sequence can be chosen. More specifically, in the case of ℓac promoter operator, such conditions can be effected by adding a suitable quantity of isopropylthio-β-D-galactopyranoside to a culture solution.
The poultry vaccine for Mycoplasma gallisepticum infections can be prepared in a manner similar to conventional technique from the thus obtained host which is concerned with the forth aspect of the invention. The host can be cultured under conditions ordinarily used for culturing microorganisms of this type. In the case of E. coli, the bacteria can be cultured in LB medium at 37°C under aerobic conditions.
After culturing, the polypeptide of the present invention can be purified by means of chromatography, precipitation by salting out, density gradient centrifugation and the like which are well known to one skilled in the art and may optionally be chosen. The thus obtained polypeptide can be used as a vaccine.
Alternatively, the host can be inactivated and the inactivated host can be used as a vaccine. In this case, the inactivation is carried out in a conventional manner after culture of the host is completed. The inactivation may be attained by heating but it is simpler to add an inactivating agent to the culture solution. As the inactivating agent, there may be used Merzonin (trademark, thimerosal manufactured by Takeda Pharmaceutical Co., Ltd.; hereinafter the same), β-propiolactone, tyrosine, salicylic acid, Crystal Violet, benzoic acid, benzetonium chloride, polymyxin, gramicidin, formalin, phenol, etc. The inactivated culture solution is added, if necessary and desired, with a suitable quantity of adjuvant. The inactivated product is then separated with a siphon or by means of centrifugation, etc. As the adjuvant, aluminum hydroxide gel, aluminum phosphate gel, calcium phosphate gel, alum, etc. are employed. The inactivated product thus separated is adjusted with phosphate buffered saline, etc. to a suitable concentration. If necessary and desired, an antiseptic is added to the product. Examples of the antiseptic which can be used include Merzonin, β-propiolactone, tyrosine, salicylic acid, Crystal Violet, benzoic acid, benzetonium chloride, polymyxin, gramicidin, formalin, phenol, etc.
In order to further enhance immune activity, adjuvant may also be added to the obtained vaccine. The adjuvant is generally used in a volume of 1 to 99 based on 100 volume of the vaccine.
When the vaccine is used, it can be mixed with diluents, filler, etc. in a conventional manner. The vaccine exhibits the effect with a dose of at least 1 µg antigenic polypeptide per kg wt. The upper limit is not critical unless the does shows acute toxicity. The dose can be determined opportunely, for example, under such conditions that the counteractive antibody titer (log₁₀) is 1.0 to 2.0. No acute toxicity was notable with a dose of 5 mg antigenic polypeptide per kg wt. to chickens.
The poultry vaccine for Mycoplasma gallisepticum infection obtained in the present invention is inoculated to poultry intramuscularly, subcutaneously or intracutaneously, etc.
Likewise the vaccine, the polypeptide of the present invention obtained by purification and isolation can be used as diagnostics since the polypeptide can strongly bind to antibody in sera collected from poultry infected with Mycoplasma gallisepticum. A test sample can be diagnosed with respect to Mycoplasma gallisepticum infections, by methods well known to one skilled in the art, such as by ELISA which comprises immobilizing the polypeptide onto a microtiter plate, reacting with poultry serum which is a test sample, then reacting with a secondary antibody labeled with peroxidase, etc. and peroxidase substrate and, determining a change in absorbancy of the reaction solution; etc.
According to the present invention, the polypeptides having antigenicity derived from Mycoplasma gallisepticum can be provided. The recombinant vectors in which DNAs encoding these polypeptides can also be provided. Furthermore, microorganisms such as bacteria, yeast, etc. transformed (or transfected) by these recombinant vectors can be provided. The polypeptides produced by these microorganisms have the same antigenicity as that of the polypeptide derived from Mycoplasma gallisepticum. Using the polypeptides, more effective vaccines and poultry diagnostics for Mycoplasma gallisepticum infection which are handled in more rapid and simpler way can be provided.
Hereafter the present invention is described in more detail by referring to the examples below. In the examples and comparative examples as well as reference examples, parts and % are all by weight, unless otherwise indicated.

Example 1

(1) Preparation of genomic DNA of Mycoplasma gallisepticum

Mycoplasma gallisepticum S6 strain was cultured at 37°C for 3 to 5 days in liquid medium obtained by supplementing 20% donor horse serum, 5% yeast extract, 1% glucose and a trace amount of phenol red as a pH indicator in 100 ml of PPLO broth basal medium. As Mycoplasma gallisepticum proliferated, pH of the culture solution decreased. At the point of time when the color of the pH indicator contained in the culture solution changed from red to yellow, incubation was terminated. The culture solution was centrifuged at 10,000 rpm for 20 minutes to collect the cells. The collected cells were then suspended in 1/10 volume of PBS based on the volume of culture solution. The suspension was again centrifuged at 10,000 rpm for 20 minutes to collect the cells. The collected cells were resuspended in 2.7 ml of PBS and SDS was added thereto in a concentration of 1%. Furthermore 10 µg of RNase was added to the mixture. The mixture was incubated at 37°C for 30 minutes to cause lysis.
The lysate was extracted 3 times with an equal volume of phenol and then 3 times with ethyl ether. The extract was precipitated with ethanol to give 200 µg of genomic DNA of Mycoplasma gallisepticum.

(2) Construction of genomic DNA library

To 40 µg of the genomic DNA of Mycoplasma gallisepticum obtained in (1) was added 4 units of restriction enzyme Aℓu I. The mixture was incubated at 37°C for 10 minutes to cause partial cleavage. The partially cleaved genomic DNA was subjected to 0.8% low melting point agarose gel electrophoresis. A DNA fragment having a length of from about 1.0 kbp to about 4.0 kbp was recovered from the gel, treated with phenol and precipitated with ethanol to give 4 µg of the DNA fragment partially cleaved with Aℓu I.
S-Adenosyl-L-methionine was added to 1.2 µg of the DNA fragment partially cleaved with Aℓu I in a final concentration of 80µM, and 20 units of EcoR I methylase was further added thereto to methylate the deoxyadenosine site in EcoR I recognition sequence, whereby the sequence was rendered insensitive to EcoR I. EcoR I linker was ligated with the DNA fragment by ligase and further mixed with a fragment of λgt11 DNA cleaved with EcoR I. Ligation was performed by ligase. Using the reaction solution, in vitro packaging was carried out in a conventional manner (DNA Cloning, vol. 1, A Practical Approach, edited by D.M. Glover). The packaging product was transfected to E. coli Y1088 (Amersham Inc.), which was then cultured at 37°C for 12 hours in LB agar medium containing 0.003% of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside and 0.03 mM of isopropylthio-β-D-galactopyranoside (IPTG). In the formed plaques, a library size was estimated by the number of white plaques and 10⁶ pfu (plaque forming unit) of DNA library was prepared.

(3) Immunoscreening of genomic DNA library

Phage obtained from the DNA library prepared in (2) was added to a suspension of E. coli Y1090 (Amersham Inc.) in 10 mM MgSO₄ aqueous solution in such a way that 500 to 1000 plaques were formed in a plate of 8 cm⌀, which was allowed to adsorb for 15 minutes. Furthermore, 2.5 ml of LB soft agar medium heated to 45°C was added and overlaid on the LB agar medium to form layers. Incubation was conducted at 42°C for 3 to 4 hours. A nylon membrane filter was immersed in 10 mM IPTG aqueous solution. After air drying, the filter was overlaid on the plate described above followed by incubation at 37°C for further 2 to 3 hours. After the incubation, the nylon membrane filter was stripped off from the plate and washed with TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). After further immersing in TBS containing 2% of skimmed milk for 30 minutes, the filter was immersed for an hour with anti-Mycoplasma chicken serum diluted with TBS to 500-fold. Thereafter, the filter was washed by immersing in TBS for 15 minutes. The filter was further washed by immersing in TBS containing 0.05% of surfactant (Tween 20) for 10 to 15 minutes. The washing procedure was repeated 4 or 5 times. Then, the filter was treated for 60 minutes with biotinated antibody to chicken IgG. After treating with a secondary antibody, the filter was washed 5 or 6 times with PBS containing 0.05% of Tween 20 and further immersed in horse radish peroxidase-avidin D solution to treat the same for 60 minutes. After the treatment, the filter was washed 5 or 6 times with PBS containing 0.05% of Tween 20 and further washed with 10 mM Tris-HCl showing pH of 8.0. Thereafter the filter was immersed in buffer containing 4-chloronaphthol and hydrogen peroxide. By this series of procedures, only the plaque in which the antigen protein derived from Mycoplasma gallisepticum had been expressed was colored to purple.
By the immunoscreening of 5 x 10⁴ plaques described above, 50 positive plaques were obtained.

(4) Preparation of immunopositive recombinant λgt11 phage DNA

E. coli Y 1090 strain was cultured at 37°C for 12 hours in LB medium containing 50 µg/ml of ampicillin and the culture solution was added to 10-fold volume of LB medium containing 10 mM MgSO₄. Then, recombinant λgt11 phage obtained in (3) which had been determined to be positive in the immunoscreening was infected to E. coli Y1090 at moi of 0.05 followed by culturing at 37°C for 5 to 10 hours. After E. coli was lysed, centrifugation was performed at 8,000 rpm for 10 minutes to obtain the supernatant. An equal volume of TM buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO₄) as well as DNase I were so added to the supernatant to show the concentration of 0.016 mg/ml followed by incubation for 15 minutes. Furthermore, NaCl and polyethylene glycol (PEG 6000) were so added thereto as to show the concentration of 0.5 M and 0.1 g/ml. The mixture was shaken at 0°C for 15 minutes. The resulting mixture was centrifuged at 10,000 rpm for 10 minutes to remove the supernatant. The obtained pellets were dissolved in 1/100 volume of TM buffer and an equal volume of chloroform was added to the mixture followed by vigorous stirring. The mixture was centrifuged at 15,000 rpm for 10 minutes and recombinant λgt11 phage was collected in the aqueous phase to obtain a phage solution.
To the phage solution were so added EDTA, SDS and pronase E as to show the concentration of 0.025M, 1%, 1 mg/ml. After incubation at 37°C for 4 hours, the solution was extracted with phenol (3 to 5 times). The extract was precipitated with ethanol to give λgt11 phage DNA containing cloned antigen DNA (M-1).

(5) Removal of the same clone from immunopositive recombinant λgt11 phage

After digesting 1 µg of each λgt11 phage DNA obtained in (4) with EcoR I, Mycoplasma gallisepticum-derived DNA was recovered by means of 0.8% low melting point agarose gel electrophoresis. The DNA was used as probe DNA.
E. coli Y1090 strain was cultured in LB medium at 37°C for 12 hours. 100 µl of the culture was mixed with 3 ml of LB soft agar medium warmed at 45°C and the mixture was overlaid on LB agar medium. On the medium 10 µl each of the respective λgt11 clones was dropped onto definite positions. When cultured at 37°C for 12 hours, plaques due to phage proliferation were formed at the dropped positions. A nylon membrane was put on this plate and the phage was adsorbed thereto. After air drying, the membrane was treated with a denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 10 minutes and further treated with a neutralizing solution (3 M sodium acetate, pH 5.5) for 10 minutes. After air drying, the membrane was heated at 80°C for 2 hours. 4-Fold SET (0.6 M NaCl, 0.08 M Tris-HCl, 4 mM EDTA, pH 7.8),10-fold Denhardt,0.1% SDS,0.1% Na₄P₂O₇,50 µg/ml of denatured salmon sperm DNA and the Mycoplasma gallisepticum-derived DNA probe which had been previously prepared and labeled in a conventional manner were added to cause hybridization at 68°C for 14 hours. After washing, the nylon membrane was overlaid on an X ray film. The presence of spot was confirmed by autoradiography. A plaque in which a spot was formed was removed as the same plaque. It was thus confirmed that 22 clones were independent.

(6) Production of recombinant plasmid (pUM1) (cf. Fig. 4)

After digesting the recombinant λgt11 phage DNA obtained in (5) with restriction enzyme EcoR I, the digestion product was subjected to 0.8% low melting point agarose gel electrophoresis. Genomic DNA fragment of Mycoplasma gallisepticum integrated into cloning site of the genomic DNA of λgt11 phage showed a length of about 0.8 kbp. The DNA fragment was extracted from the agarose gel and further extracted with phenol-chloroform (1 : 1). The extract was precipitated with ethanol and the precipitates were recovered. On the other hand, plasmid pUC18 was similarly digested with EcoR I. Then, the digestion product was extracted with phenol-chloroform. The extract was precipitated with ethanol and cleaved pUC18 was recovered. Then 5′ end phosphate was removed by treating with alkaline phosphatase. After pUC18 DNA was again extracted with phenol-chloroform, DNA was recovered by ethanol precipitation.
The cleaved pUC18 was ligated with the Mycoplasma gallisepticum genome-derived EcoR I digestion product (about 0.8 kbp) by ligase and competent E. coli TG1 strain was transformed. The transformants were cultured at 37°C for 15 hours in LB agar medium containing 0.003% of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 0.03 mM of isopropylthio-β-D-galactopyranoside and 40 µg/ml of ampicillin. Among transformed E. coli grown on the agar medium, white colonies were cultured at 37°C for 15 hours in LB liquid medium containing 40 µg/ml of ampicillin and plasmids were extracted by the method of Birnboim & Doly [Nucleic Acid Research, 7, 1513 (1979)]. After digesting with EcoR I, recombinant plasmid containing a DNA fragment having the same length of the original EcoR I fragment derived from Mycoplasma gallisepticum was detected by 0.8% low melting point agarose gel electrophoresis and named pUM1. The plasmid pUM1 is the recombinant vector of the present invention.

(7) Sequence analysis of pUM1 insert DNA

Sequence of insert DNA fragment was determined by the Dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] using pUM1 prepared in (6). A restriction enzyme cleavage map of the cloned DNA fragment is shown in Fig. 3 (a) and the nucleotide sequences of the DNA fragment are shown in Fig. 1(a)-1 and Fig. 1(a)-2.
From the facts that a molecular weight of the fused protein with β-galactosidase produced in (9) later described was about 145 killodaltons and translation termination codon (TGA) was present in bases of 706 to 708 in Fig. 1(a)-2, it is noted that the fragment encoding the polypeptide exhibiting antigenicity of Mycoplasma gallisepticum is 705 bp from 1 to 705 in Fig. 1(a)-1 and Fig. 1(a)-2 and the amino acid sequence deduced from the sequence is as shown in Fig. 1(a)-1 and Fig. 1(a)-2.

(8) Production of plasmid capable of expressing antigen protein (cf. Fig. 5)

The recombinant plasmid (pUM1) obtained in (6) was digested with EcoR I. The digestion product was subjected to 0.8% low melting point agarose gel electrophoresis. Insert DNA having a length of about 0.8 kbp was recovered from the gel and extracted with phenol-chloform. The extract was precipitated with ethanol to recover DNA.
On the other hand, plasmid pMA001 [Gene, 28, 127-132 (1984)] harboring ℓac promoter-operator and ℓac Z gene was digested with EcoR I. After the digestion product was extracted with phenol-chloroform, the extract was precipitated with ethanol and cleaved pMA001 was recovered. Then, 5′ end phosphate was removed by treating with alkaline phosphatase. After again extracting with phenol-chloroform, pMA001 DNA was recovered by ethanol precipitation.
The cleaved pMA001 was ligated with the EcoR I digestion product (about 0.8 kbp) of insert DNA by ligase and competent E. coli TG1 strain was transformed. A recombinant plasmid capable of expressing as a fused protein with β-galactosidase in which about 0.8 kbp of genomic DNA of Mycoplasma gallisepticum had been ligated in the correct direction at the downstream of ℓac Z gene of pMA001 was selected in a manner similar to (6). The recombinant plasmid was named pMAD1. This plasmid is the recombinant vector of the present invention.
Then, pMAD1 was partially digested with EcoR I. The partial digestion product was subjected to 0.8% low melting point agarose gel electrophoresis. About 7.2 kbp of fragment obtained by cleaving pMAD1 with EcoR I at one site was recovered from the agarose gel. After treatment with phenol-chloroform, cleaved pMAD1 was recovered by ethanol precipitation.
On the other hand, pSP11 [Journal of Bacteriology, June 1986, 937-944] was doubly cleaved with Hinc II and EcoR V. The cleavage product was subjected to 1.0% low melting point agarose gel electrophoresis. About 700 bp of fragment containing transcription termination sequence was recovered. After similarly treating with phenol-chloroform, the DNA fragment was recovered by ethanol precipitation. The cleaved pMAD1 and about 700 bp of DNA fragment containing transcription termination sequence were combined and cohesive ends of the respective DNA fragments were rendered blunt ends by DNA-polymerase I (Klenow's fragment). They were further ligated with ligase and competent E. coli TG1 strain was transformed. A recombinant plasmid in which the transcription termination sequence had been inserted at the downstream of genomic DNA of Mycoplasma gallisepticum of pMAD1 was selected in a manner similar to (6). The recombinant plasmid was named pMAH1. This plasmid is the recombinant vector of the present invention.

(9) Expression and detection of antigen protein (β-galactosidase fused protein)

(9-a) E. coli (MC169 strain) transformed with the recombinant plasmid obtained in (8) was precultured at 37°C overnight in LB medium containing 50 µg/ml of ampicillin. 1 ml of the preculture was taken and added to 100 ml of LB liquid medium likewise containing 50 µg/ml of ampicillin followed by culturing at 37°C. Two hours later, isopropylthio-β-D-galactopyranoside was so added thereto as to show the concentration of 1 mM followed by culturing at 37°C for further 5 hours. After the incubation, centrifugation was performed at 8,000 rpm for 10 minutes to collect E. coli. By adding 1.0 ml of PBS, E. coli was resuspended. The suspension was subjected to freezing and thawing. The cells were further sonicated and then centrifuged at 15,000 rpm for 30 minutes to recover the supernatant.
(9-b) Then, the supernatant was subjected to 8% SDS polyacrylamide gel electrophoresis (SDS-PAGE) at 50 mA for 2 hours. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 to newly detect a band of about 145 daltons. pMA001-derived β-galactosidase (in part) is approximately 115 killodaltons. It is thus considered that the newly formed polypeptide would correspond to fused protein MGg-1 in which about 30 killodaltons of polypeptide MG-1 having Mycoplasma gallisepticum-derived amino acid sequence shown in Fig. 1(a) is jointed at the C-terminus of β-galactosidase.
(9-c) On the other hand, the supernatant described above was subjected to 8% SDS-PAGE in a manner similar to (9-b) and a protein band separated in the gel was then transferred onto a nylon membrane filter. After the transfer, the filter was reacted with anti-Mycoplasma serum by procedures as in (3) to perform Western blotting. As the result, only the band corresponding to the protein of about 145 killodaltons newly found in (9-b) was detected. It was made clear that the protein was fused protein MGg-1 of Mycoplasma gallisepticum-derived antigen protein MG-1 and β-galactosidase.

Example 2

A recombinant plasmid capable of expressing Mycoplasma gallisepticum-derived antigen protein was produced from immunopositive plaque to anti-sera of Mycoplasma gallisepticum obtained in Example 1 (3), by procedures similar to Example 1 (4), (5), (6) and (8). Cloning λ phage vector used and subcloning vector and expression vector used are shown in Table 1. Restriction enzyme cleavage maps and lengths of cloned Mycoplasma gallisepticum gene are shown in Fig. 3 (b) through (v).
Then, the expression vector described above was allowed to express in a manner similar to Example 1 (9), whereby a part of β-galactosidase and a fused protein were obtained. Molecular weights of fused proteins MGg-1 to MGg-22 and antigen proteins MG-1 to MG-22 derived from Mycoplasma gallisepticum are shown in Table 1, respectively.

Example 3

Harvest of polypeptide gene TM-1 containing pUM1 polypeptide in which Mycoplasma gallisepticum has been expressed in nature

(1) Genomic Southern Hybridization of Mycoplasma gallisepticum using pUM1 insert DNA as a probe

After 1 µg of Mycoplasma gallisepticum DNA obtained in Example 1 (1) was digested with restriction enzyme EcoR I, the digestion product was subjected to 0.6% low melting point agarose gel electrophoresis. After the electrophoresis, the gel was immersed in a denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 10 minutes and further immersed in a neutralizing solution (3 M sodium acetate, pH 5.5) for 10 minutes to denature DNA. Following the neutralization, the DNA was transferred onto a nylon membrane in 6-fold SSC solution (0.7 M NaCl, 0.07 M sodium citrate, pH 7.5). After air drying, the membrane was heated at 80°C for 2 hours. 4-Fold SET (0.6 M NaCl, 0.08 M Tris-HCl, 4 mM EDTA, pH 7.8),10-fold Denhardt,0.1% SDS,0.1% Na₄P₂O₇,50 µg/ml of denatured salmon sperm DNA and pUM1 insert DNA which had been labeled in a conventional manner were added to cause hybridization at 68°C for 14 hours. The nylon membrane was overlaid on an X ray film. Autoradiography revealed that hybridization occurred on a fragment of about 1.7 kbp.

(2) Cloning of EcoR I-digested fragment of about 1.7 kbp into pUC18 and colony hybridization

After 4 µg of Mycoplasma gallisepticum DNA obtained in Example 1 (1) was digested with restriction enzyme EcoR I, the digestion product was subjected to 0.6% low melting point agarose gel electrophoresis. After the electrophoresis, a fragment of about 1.7 kbp was recovered. The fragment was ligated by ligase with pUC18 cleaved through digestion with EcoR I and competent E. coli TG1 strain was transformed. The transformants were cultured at 37°C for 15 hours in LB agar medium containing 0.003% of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 0.03 mM of isopropylthio-β-D-galactopyranoside and 40 µg/ml of ampicillin. White colonies grown on the agar medium were transferred onto a nylon membrane followed by hybridization in a manner similar to (1). Autoradiography revealed that cloning was effected and, the plasmid was named pUMGT64.

(3) Determination of the entire base sequence of TM-1

The entire base sequence of TM-1 was analyzed in a manner similar to Example 1 (7) to determine the sequence. The entire base sequence is as shown in Fig. 2(a) and 2(b).

Example 4

Production of expression plasmid pBMG6T of polypeptide TMG-1 encoded by TM-1 (Fig. 6)

Firstly, pDR540 was digested with restriction enzymes BamH I and Pst I; and the digestion product was then subjected to 0.8% low melting point agarose gel electrophoresis. A fragment was recovered from the gel. By treating with phenol-chloroform and precipitating with ethanol, a fragment of 1140 bp harboring tac promoter was recovered. Next, pUMGT64 obtained in (2) was fully digested with BamH I and then partially digested with restriction enzyme Ssp I. After 0.8% low melting point agarose gel electrophoresis, a fragment was recovered from the gel. By treating with phenol-chloroform and precipitating with ethanol, a fragment of about 840 bp containing TM-1 gene of the full length was recovered. On the other hand, pBR322 was digested with BamH I and Pst I. After 0.8% low melting point agarose gel electrophoresis, a fragment was recovered from the gel. By treating with phenol-chloroform and precipitating with ethanol, a fragment of 3240 bp was recovered. These 3 fragments were ligated with ligase and transformed competent E. coli TG1 strain. A plasmid was selected in a manner similar to Example 1 (6) and named pBMG6. This pBMG6 plasmid was cleaved with BamH I. By treating with phenol-chloroform and precipitating with ethanol, a fragment of 3240 bp was recovered. Next, 8 mer of BamH I linker was ligated with EcoR V-Hinc II fragment of about 700 bp containing the transcription termination sequence shown in Example 1 (8). By treating with phenol-chloroform and precipitating with ethanol, a DNA fragment was recovered. After the DNA fragment was digested with BamH I, the digestion product was subjected to 0.8% low melting point agarose gel electrophoresis. A fragment was recovered from the gel and treated with phenol-chloroform. By ethanol precipitation, a fragment of about 700 bp containing the transcription termination sequence was recovered. The cleaved pBMG6 and the fragment of about 700 bp were ligated with ligase. A plasmid was selected in a manner similar to Example 1 (6) and named pBMG6T.

Example 5

Expression of TMG-1

After E. coli TG1 strain transformed with pBMG6T was cultured at 37°C for 12 hours in LB medium containing 50 µg/ml ampicillin, 1 ml of the culture was collected and added to 100 ml of LB medium containing 50 µg/ml of ampicillin followed by culturing at 37°C. Two hours later, isopropylthio-β-D-galactopyranoside was so added as to show the concentration of 1 mM and culturing was continued at 37°C for further 12 hours. After culturing, E. coli was centrifuged at 8,000 rpm for 10 minutes. After the cells were collected, the cells subjected to 10% SDS-PAGE and electrophoresed at 50 mA for 2 hours. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 to newly detect a band of about 29 kilodaltons, amounting to about 10% of the total cell protein. Since this molecular weight of the protein is equal to the estimated value, said protein having about 29 kilodaltons is identified with the one encoded by TM-1 and named TMG-1.

Example 6

Purification of TMG-1

After E. coli collected in Example 5 were suspended in 10 ml of PBS, the suspension was treated by freezing and thawing and then sonicated. Then, centrifugation was performed at 8,000 rpm for 10 minutes and the supernatant was recovered. The supernatant was subjected to ion exchange chromatography (Pharmacia Fine Chemicals Inc., FPLC anion exchange column MONO Q 10/10, 20 mM triethanolamine, pH 7.3, NaCl density gradient 0 M to 1 M [60 minutes], sample amount of 20 mg, flow rate of 4 ml/min, fraction size of 4 ml). From each of the collected fractions, 10 µl was adsorbed onto a nitrocellulose membrane. Immuno dot blotting (primary antibody; Mycoplasma gallisepticum infected chicken serum, secondary antibody;biotinated anti-chicken IgG rabbit serum, color forming system;chicken peroxidase complex, substrate;4-chloro-1-naphthol) was carried out, whereby TM-1 polypeptide could be detected around 0.6 M of NaCl concentration. The detected fraction was subjected to 8% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. It was confirmed that about 90% of the total protein was TMG-1. By the procedures, about 200 µg of TMG-1 could be purified from the culture solution of TG1 transformed by pBMG6T.

Example 7

Test on growth inhibition of Mycoplasma gallisepticum

Polypeptide MGg-1 encoded by pMAH1 which was obtained in a manner similar to Example 2 and TMG-1 obtained in Example 6 were respectively dissolved in Dulbecco's PBS buffer so that each shows the concentration of 500 µg/ml. Each solution, 1 ml, was subcutaneously injected to each Japanese white rabbit weighing about 2 kg, together with an equal volume of complete Freund adjuvant respectively. Further 4 weeks later, each of MGg-1 solution, 0.5 ml, and TMG-1 solution, 0.5 ml, described above was respectively administered to each rabbit subcutaneously as well as intravenously into the ear vein for the second immunization. Seven days later, it was confirmed in a conventional manner that antibody titer was increased and, anti-MGg-1 serum and anti-TMG-1 serum were collected from the ear artery of the rabbit.
On the other hand, Mycoplasma gallisepticum was liquid cultured by the method described in Example 1 (1). The culture solution, which color changed from red to yellow by a pH indicator, was diluted to 128-fold with medium for Mycoplasma gallisepticum. To 500 µl of the diluted culture solution was aseptically added 25 µl each of 6 samples: Mycoplasma gallisepticum infected chicken serum, the MGg-1 rabbit serum and anti-TMG-1 rabbit serum, standard rabbit serum and standard chicken serum which were quite free of antibody to Mycoplasma gallisepticum and medium for Mycoplasma gallisepticum. By culturing at 37°C, growth inhibition test was carried out.
On Days 0, 3 and 4 of the incubation, 10 µl each was collected from each of the culture solutions for growth inhibition test of Mycoplasma gallisepticum. Each of the collected culture solutions was spread on a plate of 1.2% agar medium for Mycoplasma gallisepticum followed by culturing at 37°C in a 5% CO₂ incubator. The number of cells in the corresponding culture solution was deduced from the number of colonies of Mycoplasma gallisepticum formed, 7 days later. The results are shown in Table 2.

When the added sample was the culture solution of standard rabbit serum, standard chicken serum or medium, there was no difference in a proliferation rate of Mycoplasma gallisepticum and, the cell number reached the saturation on Day 3 of the incubation. In the culture solution added with anti-MGg-1 rabbit serum, anti-TMG-1 rabbit serum and infected chicken serum, proliferation of Mycoplasma gallisepticum was not proliferated at all up to Day 3. The results reveal that TMG-1 and fused proteins MGg-1 are antigens capable of inducing antibodies which effectively inhibit the growth of Mycoplasma gallisepticum.

Table 2

Sample	Day	0*	Day 3	Day 4
Anti-MG-1 rabbit serum	9.8 x 10³	1.1 x 10⁴	1.9 x 10⁴
Anti-TMG-1 rabbit serum	9.8 x 10³	1.0 x 10⁴	9.0 x 10³
Standard rabbit serum	9.8 x 10³	2.8 x 10⁶	3.3 x 10⁶
Infected chicken serum	9.8 x 10³	1.0 x 10⁴	1.3 x 10⁴
Standard chicken serum	9.8 x 10³	2.9 x 10⁶	3.2 x 10⁶
Culture medium	9.8 x 10³	3.0 x 10⁶	3.2 x 10⁶

* The day when the incubation was started.

Numerical values in the table indicate the number of cells in 1 ml of the culture solution.

Example 8

E. coli transformed by pMAD1 was cultured in LB medium at 37°C for 24 hours. The culture solution, 10 ml, was added to 1 ℓ of LB medium charged in Sakaguchi flask followed by culturing at 37°C for 2 hours. Isopropylthio-β-D-galactopyranoside was added thereto in a concentration of 1 mM followed by culturing for 24 hours. The culture solution was centrifuged at 5,000 rpm for 10 minutes while keeping at 2 to 5°C. After the precipitates were centrifuged and washed with phosphate buffered saline (pH 7.0) 3 times, 10 ml of phosphate buffered saline containing 0.01% of thimerosal was added to the precipitates. After sonication was performed for 30 seconds 5 times, the supernatant was collected and made crude antigen. To 10 volume of the crude antigen were added 10 volume of aluminum hydroxide gel containing 0.01% of thimerosal and 30 volume of sterilized phosphate buffered saline. The resulting mixture was made a vaccine.
The vaccine described above was intramuscularly inoculated to 3 SPF chickens at the age of 17 days in a dose of 0.5 ml. For supplemental immunization, the vaccine was intramuscularly inoculated at the age of 39 days. At the age of 66 days, agglutination reaction test of Mycoplasma gallicepticum and hemagglutination inhibition (HI) test were carried out to determine immune effect.
In the agglutination reaction test, 0.05 ml of Mycoplasma antigen manufactured by Nippon Pharmacy Co., Ltd. and 0.05 ml of vaccinized or non-vaccinized chicken serum were mixed on a glass plate and those forming a clear agglutination mass within 2 minutes were determined positive.
The HI test was performed by adding an equal volume of culture solution of 4 units of Mycoplasma gallisepticum S6 strain to each of diluted sera, mixing them, settling the mixture for 10 minutes to sufficiently sensitize and then adding blood cells thereto.
The results of the cell agglutination reaction test and HI test are shown in Table 3. The vaccinized chicken serum according to the present invention showed a markedly increased agglutination effect, indicating that the vaccine of the present invention exhibits highly immunizing effect.

Example 9

Diagnosis of poultry Mycoplasma infection

Polypeptide obtained in Run No. (2-1) of Example 2 and TMG-1 obtained in Example 6 were dissolved in bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃), respectively, in concentrations of 10 to 100 µg/ml.
On the other hand, bicarbonate buffer in which 50 µl/well of the polypeptide had been dissolved was charged in a 96 well microtiter plate. After allowing to stand at 4°C overnight, incubation was carried out at 37°C for an hour to immobilize. After the immobilization, the plate was washed 5 times with 1% bovine serum albumin-containing PBS. To each well of the microtiter plate was added 100 µl each of sera collected from chickens infected with Mycoplasma gallisepticum as a positive control and sera collected from test chickens. Then incubation was performed at 37°C for an hour. Each well was washed 5 times with PBS. A 1000-fold diluted solution of rabbit IgG which bound horse radish peroxidase to chicken IgG was added to each well in an amount of 100 µl each per well followed by incubation at 37°C for an hour. After again washing with PBS 5 times, 2,2′-azino-di-[3-ethyl-benzthiazoline sulfonate] which was substrate of horse radish peroxidase was added followed by incubation at 37°C for 30 minutes. Absorbance was measured at a wavelength of 405 nm using an immunoreader. As the result, only the serum collected from the chicken infected with Mycoplasma gallisepticum selectively absorbed the wavelength of 405 nm. The results reveal that the polypeptide can be utilized as a poultry diagnostic for Mycoplasma gallisepticum infection.

Claims

A polypeptide capable of reacting with anti-Mycoplasma gallisepticum poultry sera through an antigen-antibody reaction, said polypeptide being (i) encoded by a DNA fragment which has one of the restriction enzyme cleavage maps shown in Figs. 3-1, 3-2, 3-3 and 3-4, or (ii) a polypeptide having substantially the same capability of reacting with anti-Mycoplasma gallisepticum poultry sera as a polypeptide according to (i).
A polypeptide as claimed in claim 1, wherein said polypeptide is MG-1 whose amino acid sequence is described in Fig. 1(a)-1 and Fig. 1(a)-2, MG-2 whose amino acid sequence is described in Fig. 1(b), MG-3 whose amino acid sequence is described in Fig. 1(c), MG-4 whose amino acid sequence is described in Fig. 1(d), MG-5 whose amino acid sequence is described in Fig. 1(e), or MG-6 whose amino acid sequence is described in Fig. 1(f)-1 and Fig. 1(f)-2.
A polypeptide which has the same amino acid sequence as that of a polypeptide expressed in Mycoplasma gallisepticum in nature and which comprises the amino acid sequence of a polypeptide of claim 1 or claim 2.
A polypeptide as claimed in claim 3, wherein said polypeptide is TMG-1 having an amino acid sequence shown in Fig. 2.
A fusion protein capable of reacting with anti-Mycoplasma gallisepticum poultry sera through an antigen-antibody reaction, whose C-terminus is a polypeptide according to claim 1 and whose N-terminus is an enzyme protein derived from bacteria.
A fusion protein as claimed in claim 5, wherein said enzyme protein derived from bacteria is β-galactosidase or β-lactamase.
A DNA fragment encoding a polypeptide as claimed in any one of claim 1 through 4.
A DNA fragment according to claim 7 which is derived from Mycoplasma gallisepticum having a restriction enzyme site map and length shown in any one of Fig. (3) (a) through (v).
A DNA fragment according to claim 7, which base sequence is selected from the sequences described in Fig. 1(a)-1 through Fig. 1(f)-2.
A DNA fragment according to claim 7, wherein the base sequence is described in Fig. 2.
A recombinant vector having integrated therein a DNA fragment according to any one of claims 7 to 10.
A host transformed by a recombinant vector of claim 11.
A poultry diagnostic for Mycoplasma gallisepticum infection comprising a polypeptide or fusion protein as claimed in any one of claims 1 to 6 as an effective ingredient.
A poultry vaccine for Mycoplasma gallisepticum infection comprising a polypeptide or fusion protein as claimed in any one of claims 1 to 6 as an effective ingredient.
A poultry vaccine for Mycoplasma gallisepticum infection according to claim 14, wherein said polypeptide is MG-1, TMG-1 or a polypeptide having substantially the same capability of reacting with anti-Mycoplasma gallisepticum poultry sera as MG-1 or TMG-1.